PROJECT SUMMARY/ABSTRACT The five-year survival rate of cancer patients has improved in the last thirty years. The survival rate of pediatric cancers has reached 80%, and the young cancer survivors go on to live generally normal lives due to their life- saving treatments. Although advances in cancer therapies increased the survival rate, these therapies have long-term adverse health effects. One of the most serious side effects of those therapies is the off-target effect on germ cells. It causes the loss of primordial follicles that consist of ovarian reserve, which is defined as all of the follicles in the ovary available for future fertility and endocrine support for women. This treatment results in premature ovarian insufficiency, which clinically presents as endocrine dysfunction and infertility. Prepubertal kids who are treated with cancer therapies cannot initiate puberty and need medical help for maintaining their normal life. Thus, developing an effective intervention is an unmet need in the field. It has been proposed that certain intervention agents have the efficacy to protect ovarian reserves from cancer therapies. However, the mechanisms underlying the fertoprotective effects of these adjuvant therapies remain largely speculative. Therefore, our goals are to clarify the mechanisms on how gonadotoxic therapies deplete primordial follicles in the ovarian reserve, develop novel fertoprotective agents based on the mechanisms of primordial follicle depletion, and mechanistically match adjuvant fertoprotective strategies with specific gonadotoxic treatments. Several theories have been proposed to explain the mechanism of primordial follicle loss. The burn out theory is that chemotherapeutic agents activate dormant primordial follicles through an activation pathway. Another theory is that chemotherapeutic agents destroy primordial follicles through an ?apoptotic pathway? due to high sensitivity to DNA damage. Our preliminary data suggest that there are two distinct apoptotic pathways in oocyte death in primordial follicles by DNA damages. Guided by strongly supported preliminary data, we propose to test 6 common chemotherapeutic agents and signaling pathway-based inhibitors (1) to elucidate the mechanisms of ovarian reserve depletion by 6 common gonadotoxic agents, (2) to elucidate the cellular mechanism of primordial follicle depletion by gonadotoxic agents in genetically modified mouse models, and (3) to examine the efficacy and safety of ovarian reserve-protecting adjuvant therapies. Our proposed studies will have a significant impact on the field by (1) clarifying mechanisms by which specific chemotherapeutic agents deplete primordial follicles, and by (2) testing the preclinical fertoprotective efficacy and safety of candidate adjuvants against specific chemotherapeutic agents. Our studies may inform guidelines for strategic selection of fertoprotective agents based on the mechanism of action against common gonadotoxic agents to prepubertal kids who will be treated with cancer therapies.